The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Oxygen and silicon are the most and second most abundant elements, respectively, in the earth's crust and consequently a great deal of mass of the Earth's crust is silica (59%). Silica (silicon dioxide, SiO2) is a versatile material that is utilised in great number of applications, such as glass preparation, casting moulds, additive in concrete & cement, pesticides, fertilisers, catalysis, chromatography columns, as xerogel dryer material in desiccators, as aerogel in space dust collection, functional filler in paints or sunscreens, water filtration, drug and gene delivery.
Silica can be obtained from naturals sources by mining and desired modification or by synthesizing it from various precursors. Industrial sand and gravel obtained by mining, often called “silica,” “silica sand,” and “quartz sand,” includes sands and gravels with high silicon dioxide (SiO2) content. Silica can also be prepared/modified to many different structures by fuming or wet synthesis methods, which results in different properties both with respect to textural features and (surface) chemistry.
One of the most interesting features of silica is its interaction with many living organisms and biomolecules. Certain crystal forms of silica are harmful, as in the case of silicosis (“Coal worker's disease”), where inhaled crystalline silica accelerates fibrous tissue overgrowth in lungs, but in amorphous and water-dissolved form silica has been observed to have positive interaction with living organisms and biomolecules. There is also a living example in nature showing the potential of silica for natural contact with living organisms, an example on the phenomenon that is sometimes called biosilicification that results in biogenic silica. One of the most abundant living creatures on earth, diatoms, use the corresponding wet synthesis method to prepare a silica “skeleton” to cover its organic part, i.e., diatoms induce the synthesis of amorphous silica by extracting the needed soluble silica, silicic acid, from sea water that nucleates and condensates on diatoms creating a silica surface.
One of the most studied methods to prepare silica is the sol-gel method. It is done in liquid phase, which makes it potential for many applications. Sol-gel derived SiO2 and other SiO2-based materials are commonly prepared from alkoxides, alkylalkoxides, aminoalkoxides or inorganic silicates that via hydrolysis form a sol that contains either partly hydrolysed silica species and/or fully hydrolysed silicic acid. Consequent condensation reactions of SiOH containing species lead to formation of larger silica species with increasing amount of siloxane bonds. Furthermore, the species aggregate, form nanosized particles and/or larger aggregates until a gel is formed. Reactions (typically at ≦40° C.) are commonly catalysed either by mineral acids (such as HCl and HNO3) or bases (such as NH3). The formed gel is then aged (typically at ≦40° C.), dried to different water content (typically at ≦40° C.) and/or heat-treated (typically at ≦700° C.) to desired form resulting typically in amorphous and porous SiO2. The last step, heat treatment at elevated temperatures (50-700° C.) is typically skipped if the system contains a biologically active agent. The gels that are dried at moderate temperature (at ≦50° C.) are called xerogels (<Gr. xero=dry). The silica gels containing substantial amounts of water, e.g. 30-90%, are sometimes called hydrogels. Amorphous and porous sol-gel derived SiO2 is known to be biocompatible and known to dissolve in the living tissue as well as in solutions simulating the inorganic part of real human body fluid, e.g. in a water solution buffered to pH 7.4 at 37° C. with or without inorganic salts found in real body fluids.
Amorphous silica made by the sol-gel method is known to result in nanoscale porous structure with varying amount of hydroxyl groups on surface. It resembles silica structures formed in biosilicification processes. Such silica has been observed to have specific interaction with living organisms and many biomolecules. It is known to be biocompatible, (e.g., acceptable response observed in tissue) and adjustably biodegradable in simulated physiological conditions and in living tissue. Consequently, sol-gel derived silica and other amorphous silica-based materials are also used as such in biomaterials applications and tissue engineering. Due to possibility for easy encapsulation of different molecules and other active agents by adding them into the reacting sol in liquid phase, silica has also been used as drug delivery device for traditional small-molecule drugs and different biologically and therapeutically active agents, such as proteins and viral vectors.
Encapsulation can also be utilized in many other applications. Many proteins and enzymes are useful in (bio)catalysis or in diagnostic applications as sensors (e.g., antibody-antigen) and they can be encapsulated in sol-gel derived silica, which acts as a carrier material. Also living cells can be encapsulated in silica, where they may act as bioreactors, e.g., by producing therapeutic proteins. Hence, studies on preservation of the biological activity of proteins and other active agents in silica have been one of the topics of interest in different fields of science. In addition to sensitive agents in different biotechnology-related applications, it is also possible to encapsulate other active molecules, which are usually easier cases with respect to preservation of their activity and functionality, such as antimicrobial agents, fragrances, perfumes, colours & dyes, food colours, food additives, antioxidants, humidifiers, vitamins, explosives, insecticides, herbicides, fungicides and high-price reagents/precursors for chemical reactions.
Molecules and other active agents encapsulated in sol-gel silica are in direct contact with different silica species from the liquid phase sol to solid-phase dominating gel, where the condensation and pore structure are under continuous development. Quite substantial shrinkage may occur during the aging and drying processes and also chemical reactions, such as condensation, proceed, so the structure, aging and drying during the storage, may have crucial effects on the activity of the encapsulated agents.
Storage in water-based solution provides a natural and favourable water surrounding to biomolecules, living cells, viruses and other active agents. Drying of sol gel derived silica gel causes shrinkage of the nanoscale pore structure and also results in the formation of a brittle surface structure. Silica is used in many form, such as monoliths, fibres, particles of different size and coatings. The potential applications of fibres are usually composed of structures where silica-silica interaction is present on a macroscopic scale, e.g., in fibre nets or other corresponding structures. In such cases, drying of the silica structure makes the structure loose its flexibility, which is an important property, e.g. in surgical use, e.g., implantation. Some silica formulations may also crack when dried and then replaced in water, e.g. after implantation into living tissue containing a substantial amount of water. This is because the shrinkage of the pore structure during drying is an irreversible process and replacement in water is followed by immediate diffusion of water into the nanoscale pores causing high stresses through capillary forces.
The loss of the activity or viability of proteins, enzymes, viruses, bacteria and other cells in the sol-gel derived silica has been discussed in many publications. The sensitivity to surrounding conditions varies depending on specific agents and there might be differences within species also, e.g., between different proteins. Aging, drying, surface structure, the role of water and alcohol amount in the structure are examples on the factors that may have an influence. One of the major concerns is the long-term stability of biological activity and/or viability of the encapsulated agents as well as the possible breakage of the materials.
Wang et al. [R. Wang, U. Narang, P. N. Prasad and V. Bright, Affinity of antifluorescein antibodies encapsulated within a transparent sol-gel glass, Anal. Chem., 65 (1993) p. 2671-2675] discloses encapsulation of an antibody in a sol-gel matrix and storage of the matrix encapsulating the antibody in water for up to 5 weeks. The sol-gel matrix was stored in water to retain affinity of the antibody.
Livage et al. (J. Livage, T. Choradin and C. Roux, Encapsulation of biomolecules in silica gels, Journal of Physics: Condensed Matter, 13 (2001) p. R673) discussed that sol-gel derived silica is potential for encapsulation of biomolecules and that the internal water in silica has an influence on protein folding, but they also comment that the long term stability of the encapsulation systems should be addressed fully.
N. Nassif et al. (N. Nassif, O. Bouvet, M. N. Rager, C. Roux, T. Choradin, J. Livage, Living Bacteria in silica gels, Nature Materials, vol 1 (2002) p. 42) showed prolonged viability of bacteria in silica gels, but they comment that the long-term viability of cell in inorganics has never been fully addressed.
I. Gill & A. Ballesteros (I. Gill, Bio-doped Nanocomposite Polymers: Sol-Gel Bioencapsulates, Chemistry of Materials 13 (2001) p. 3404 and I. Gill & A. Ballesteros, Bioencapsulation within Synthetic Polymers, TIBTECH vol. 18 (2000) p. 282)) have also reviewed extensively the encapsulation of biomolecules in different silica gels, both xerogels and hydrogels (gels with 50-80% interstitial water and pore sizes between 4-200 nm). However, they address that the hydrogels are usually more or less dried and the structural collapse and shrinkage and a consequent loss of biological activity and breakage of the silica gel structure are common.
Zusman et al. (Zusman et al., Purification of sheep immunoglobin G using protein A trapped in sol-gel glass, Analytical Biochemistry, 201 (1992) p. 103) have also shown that an intact influenza virus entered a sol-gel derived silica gel with high water content (a hydrogel), where it could react with the previously encapsulated sialic-acid-coated liposome indicating thus also indirectly the preservation of the biological activity of the virus during the stay in the silica gel.
M. L. Ferrer et al (M. L. Ferrer, L. Yuste, F. Rojo, F. del Monte, Biocompatible sol-gel route for encapsulation of living bacteria in organically modified silica matrixes, Chemistry of Materials, 15 (2003) p. 3614) concluded in their studies on encapsulation of living bacteria that in addition to alcohol-free system, the main factor affecting the solubility of the encapsulated bacterial cells in the sol-gel silica materials is the control of the physical constraint of exerted by the silica matrix (syneresis) during the aging, i.e., they suspect that the shrinking structure during the aging and drying may reduce the viability of cells.
K. K. Flora and J. D. Brennan (K. K. Flora and J. D. Brennan, Effect of Matrix Aging on the Behavior of Human Serum Albumin Entrapped in a Tetraethyl Orthosilicate-Derived Glass, Chemistry of Materials 13 (2001) p. 4170) have studied albumin encapsulation in TEOS-derived silica gels in different conditions (dry-aged (in air without washing), wash-aged (drying in air after washing) and wet-aged (in buffer) and they concluded that regardless of the method, after 2 months of aging, the entrapped proteins retained less than 15% of their ligand binding ability in solution due to partial unfolding. However, they studied the situation inside the gel only and for albumin only and they did not take into account that the (partial) unfolding, often caused by adsorption to silica pore wall surface or by extensive aggregation of proteins, has been observed to be reversible for many proteins, e.g., it varies depending on the case and this reversible process is also possible as the proteins are desorbed as they are released. Although the structure is shrinking during the aging (independently whether the drying is allowed or not), the simultaneous aging and drying is more effective and the structure may also reach the point where the encapsulated agents do not only adsorb differently, but they may also be physically squeezed in the pores and this occurs naturally faster for larger agents, like cells and viruses. In other words, although the loss of bioactivity is observed inside silica, it may be reversible, but upon too extensive drying, it may become irreversible.
T. Wilson et al. (T. Wilson, R. Viitala, H. Jalonen, R. Penttinen, and M. Jokinen, The Release and Biological Activity of Lysozyme from Selected Sol-gel Derived SiO2 Matrices, submitted to Journal of Materials Science: Materials in Medicine, January 2007) observed that a sol-gel derived silica gel, which had not dried, preserved the activity of released lysozyme, i.e., the amount of biologically active lysozyme corresponded almost 100% to the total amount of released lysozyme with or without separate protective agent, but the activity of released lysozyme was substantially reduced already within few weeks as the gel was let to dry and age simultaneously, i.e., the loss of bioactivity had become irreversible.
D. Avnir et al. (D. Avnir et al., Enzymes and other Proteins Entrapped in Sol-Gel Materials, Chemistry of Materials 6 (1994) p. 1605) reviewed extensively the encapsulation of enzymes, whole cells, antibodies and other proteins in sol-gel materials. They mention wet gels and xerogels, but they do not mention the storage of silica in aqueous solutions, but rather the problems, such as brittleness of the structure of the dried, glassy silica matrix. They are also mostly interested in biological activity in situ in silica gel, where already a reversible loss of biological activity is to be avoided.
M. Jokinen et al. (WO 2005/082781) stored silica implants with encapsulated drugs and other biologically active agents in buffers saturated with silica's dissolution products in order to compare the differences in release mechanisms with an in sink dissolution system, where the silica was let to dissolve freely. The preservation of biological activity or other functional properties of silica or the encapsulated agents were not studied.
P. Ducheyne et al. (U.S. Pat. No. 5,874,109) immersed silica-based implants in aqueous buffer solutions to study the release of encapsulated drugs. This represents a typical and commonly used example in studying silica-based biomaterials where materials are immersed in different buffer solutions mimicking, at least partly, the contents & properties of body fluids to simulate their properties, e.g., ability to precipitate bone mineral-like calcium phosphate (hydroxyapatite), biodegradation property or release of encapsulated agents in vitro.